Analyzing Unionicola Phylogeny II:

The mollusk mites

Malcolm F. Vidrine and Dale D. Edwards

The detailed analyses that involves the content in this work is provided in our book entitled Mites of Freshwater Mollusks (Edwards and Vidrine 2013—the book is out of print, but copies are available at several locations online (and if you need a digital copy, we will be happy to send one via email in a Dropbox® account)). Our goal here is to provide a short discussion that is simple and easy to translate internationally.

Mites commonly occurring in freshwater mollusks and sponges are diverse and in many ways poorly understood. One group, Unionicola (Unionicolidae: Unionicolinae), has however received considerable attention. While molecular phylogenetic analysis is at its infancy, several morphological studies, including the genus-wide analyses by us (Edwards and Vidrine 2013), have been conducted. Three major studies (Edwards and Vidrine 2006, Edwards et al. 2010 and Wu et al. 2009, 2012) initiated the re-evaluation of the phylogeny of the genus, but these studies were limited to 8-11 subgenera each. The genus contains 57 subgenera, with approximately more than half of both the subgenera and the species known to be associated with mollusks—a rather unusual lifestyle.

Some basic questions and our answers (in red) based upon the trees:

Does the genus Unionicola appear to be a single evolutionary unit (clade)? Yes

Do the subgeneric categories as established reflect the implied phylogeny of the analyses? Yes

Are the mollusk mites represented as a separate phylogenetic unit? Yes

Do biogeographic range distributions of the mites appear to support or refute the findings of the analyses? Support

Do host range distributions of the mites appear to support or refute the findings of the analyses? Support

Do behavioral range distributions, e.g., mantle mites vs gill mites, of the mites appear to support or refute the findings of the analyses? Support

Text from Edwards and Vidrine (2013) modified and updated:

The origin and evolutionary diversification of the unionicolid mites is not well understood and what little information is available has been derived mainly for the gill mites based on their geographical distributions and patterns of variation in a few morphological characters. The most obvious presentation in the tree is the separation of the mollusk-associated mites into groups, both gill mites and mantle mites. While there is one major group of mantle mites, there are 3 groups of gill mites: Southern, American and AfroEurAsian groups.

Vidrine and Cordes (2004) suggested that the least-derived group of gill mites were species of the subgenus Kovietsatax, and that these mites were morphologically similar to free-swimming mites of the subgenus Downesatax. Species of the subgenus Kovietsatax are found in mussels of the family Hyriidae (mussels from the order Unionoida native to South America, Australia, New Zealand and New Guinea) from Australia and appear to be ancestral to mites of the subgenus Hyricola, another group of gill mites found in hyriid mussels of Australia. The hypothesis that Kovietsatax mites represent the ancestral group to all Unionicola gill mite subgenera (Vidrine and Cordes, 2004) is further substantiated by the fact that their host mussels (hyriids) are ancestral to freshwater mussels of the family Mulleriidae from Central and South America and Unionid mussels from Africa, Asia, and North America (Graf and Cummings, 2006; Hoeh et al., 2009). This group is considered the most ancient, and for convenience, we labelled it as the Southern (Austral) group.

Two mite subgenera, Atacella which occur with South American mussels of the families Etheriidae, Hyriidae and Mulleriidae, and Coelaturicola, an African taxon from mussels of the families Unionidae and Iridinidae, are, based on anatomical characteristics, thought to be closely related to mites from the subgenus Hyricola (Vidrine et al., 2007a; Vidrine

et al. 2007b), thus representing early radiations of gill mites on these two continents. In South and Central America, seven subgenera of gill mites (Atacella, Australatax, Berezatax, Ferradasatax, Polyatacides, Unionicolella, and Unionicolides) are recognized and morphologically these mites form a distinct group of gill mites (Vidrine et al., 2008). Mites from the subgenera Berezatax and Unionicolides also appear in North America, with the latter subgenus boasting more species in North America than in Central and South America combined (Gledhill and Vidrine, 2002). Interestingly, Unionicolides gill mites from North America occur in association mussels of the subfamily Ambleminae, one of the most diverse lineages of freshwater mussels in the world (Vidrine, 1986c). This group of derived mites is here labelled as the American group.

African gill mites include species from the subgenera Coelaturicola, Mutelicola, Iridinicola, and Chambardicola. Coelaturicola mites are morphologically intermediate between mites from the subgenera Mutelicola and Hyricola and are interpreted as being the ancestral lineage of gill mites from Africa (Vidrine et al., 2007b). Many of the morphological characters among African gill mites show intermediate states between Hyricola from Australia and Prasadatax gill mites from India, prompting Vidrine and Cordes (2004) to suggest that gills mites from Australia underwent a radiation in Africa followed by a radiation in India. One subgenus of gill mites (Prasadatax) occurs in India. Mites of the subgenus Prasadatax are morphologically intermediate between African gill mites and Asian and European gill mites from three subgenera Unionicola, Dimockatax and Wolcottatax (and possibly Fulleratax) (Vidrine, 1992). The latter three subgenera also occur in association with freshwater mussels from North America. These gill mites from Africa, Europe, Asia and North America are labelled the AfroEurAsian group. Some of the species are a common part of the North American fauna, but there are no subgenera unique to North America—thus these appear to be extensions in range, both geographic and host ranges, into North America from Eurasia and maybe Africa. Mantle mites, in part, appear to be derived from this group of gill mites.

A phylogenetic analysis of Unionicola mites based on molecular sequence data would undoubtedly be the best approach to resolve evolutionary relationships among taxa that comprise the genus. There are,

however, at least two compelling reasons why generating a molecular phylogeny for the group could present difficulties and thus warrant reconstructing the evolution history among these mites based on morphological data. First, many of the mollusk mites that have been identified and described were collected long ago and would be difficult to recollect primarily due to host extinction and habitat destruction. Second, holotypes and representative paratypes of described species have been preserved in solutions that have invariably damaged the quality and integrity of their DNA. Unfortunately, many of the described species of Unionicola may be difficult to recollect primarily due to host extinction and habitat destruction. In short, a phylogenetic analysis of Unionicola mites based on non-molecular data would presumably allow for greater taxon sampling.

Addressing evolutionary relationships among Unionicola mites based on morphological criteria is not without its challenges, given that so few characters historically have been used to diagnose the genus and its subgenera (Cook, 1974). Also, a cursory glance at the taxonomic studies involving Unionicola mites suggests that a limited number of characters are available for phylogenetic inference. It should, however, be noted that there have been no previous attempts to diagnose subgenera and species using a large number of morphological characters and to subsequently address patterns of variability of these characters across taxa. We recently went through the taxonomic literature for Unionicola mites and generated 158 characters (157 morphological characters and one life history character) that could be used to estimate evolutionary relationships among the currently named subgenera (N=57) that comprise the genus, especially relationships between free-swimming taxa and those that have adopted symbiotic lifestyles. We also identified 139 characters to reassess and potentially resolve conflicting hypotheses regarding the phylogeny of Unionicola mites that occur in association with mollusks (molluscan gill mites and mantle mites).

In conclusion, the results of these analyses suggest that the mollusk mites represent a monophyletic clade. In addition, they suggest that the mantle mites are a sister taxon to the African and Eurasian gill mites. A close affinity between mantle mites and gill mites was also indicated by

Edwards et al. (2010) in their paper assessing evolutionary relationships among molluscan-mite subgenera of North America. Interestingly, the phylogenetic hypothesis for the Unionicola mollusk mites, especially the gill mites, appears to dovetail our present understanding of the diversification of these mites. The Australian mites (in particular the subgenera Hyricola and Kovietsatax) are thought to represent the least derived group of gill mites and these mites are the first clade to branch in the proposed tree for mollusca mite taxa. In the mollusk-mite tree the South American (Atacella, Australatax, Unionicolides, and Unionicollela) and North American (Berzatax and Unionicolides) gill mites form a distinct clade from a monophyletic grouping that includes African gill mites (Chambardicola, Coelturicola, Iridinicola, and Mutelicola) and mantle mites. These grouping are consistent with the hypothesis that mites on South American and African continents represent early radiations of Unionicola mites from an ancestral stock occurring in Australia. Mites from the subgenus Unionicolides occur in both the South American and North American continents and their occurrence in North America appears to represent a secondary radiation that coincides with the diverse radiation of their host mussels on this continent. Mites from subgenus Prasadatax from India appear to have characteristics that are shared by African gill mites and many of the mite subgenera from Eurasia (Dimockatax, Fulleratax, Unionicola, and Wolcottatax). These African and Eurasian mites occur largely as a distinct clade of the tree. Not surprisingly, the three subgenera (Dimockatax, Unionicola, and Wolcottatax) that occur in both Eurasia and North American form a monophyletic grouping. Vidrine argued that mites from these North American subgenera represent descendant lineages from the Eurasian continent. Again, a phylogenetic hypothesis for these mites based on molecular sequence data should be done to confirm the validity of these evolutionary scenarios.

A robust phylogeny of Unionicola mollusk mites based on molecular sequence data potentially can be examined in the context of the phylogenetic history of their host mussels (Roe and Hoeh, 2003; Graf and Cummings, 2006; Hoeh et al., 2009), an avenue of research that largely has been unexplored. The information that is available is largely speculative or peripheral to cophylogenetic studies. Vidrine (1996b)

suggested that the evolution of unionicolid mussel-mites is closely tied to the evolutionary history of their hosts, given that major clades of mussels harbor unique assemblages of Unionicola subgenera. Edwards and Vidrine (2006) found significant difference in host specificity among Unionicola gill mites when compared to mantle mites and argued that gill mites were more specialized because of the long evolutionary history gill mites have had with host mussels (Vidrine et al., 2005).

Cryptic species within currently accepted species, e. g., U. hoesei, U. arcuata and U. minor, may well exemplify the nature of species taxa in the genus. With molecular analyses, we are on the verge of beginning to understand not only the true diversity within the genus but also the nature of the coevolution of mollusks and mites.

Figure 8.5 Bayesian tree based on 139 morphological characters for representative species from 30 subgenera of Unionicola mollusk-associated mites. Subgeneric designations for the representative species presented in the tree are indicated in parentheses. (figure from Edwards and Vidrine 2013)

Designations denoted by letters

A. Designates the first group (Southern) gill mites from Australia and South America. These appear to be the least derived of the mollusk-associated mites.

B. Designates the mollusk-associated mites other than the gill mites in A.

C. Designates the mollusk-associated mites known as the second group (American) of gill mites from South America and North America.

D. Designates the mantle and gill mites not in C. E. Designates the mollusk-associated mites known as mantle mites.F. Designates the mollusk-associated mites known as the third group

(AfroEurAsian) of gill mites from Africa, Europe, Asia and North America.

Comments

1. Unionicolopsis (only females) apparently provides little information. There is little doubt that it is distinctive, and it may represent a separate genus as indicated in its description.

2. The gill mites break into 3 groups: Southern (A), American (C) and AfroEurAsian (F).

3. The Southern group of gill mites includes the subgenera Kovietsatax and Hyricola. These infect mussels in the family Hyriidae in Australia.

4. The American group of gill mites includes the subgenera Atacella, Polyatacides, Australatax, Berezatax, Ferradasatax, Unionicolella and Unionicolides. They are distributed across North and South America in a variety of mussels including members of Unionidae, Etheriidae, Mulleriidae and Hyriidae.

5. The AfroEurAsian group of gill mites includes the subgenera Coelaturicola, Iridinicola, Chambardicola, Mutelicola, Unionicola, Dimockatax, Wolcottatax, Prasadatax and Fulleratax. They are distributed across Africa, Europe, Asia and North America in a variety of mussels including members of Unionidae and Iridinidae.

6. The mantle mites include the subgenera Baderatax, Ampullariatax, Polyatax, Imamuratax, Neoatax, Causeyatax, Breaudatax, Clarkatax, Pentatax, Anodontinatax, Vidrinatax, Vietsatax and Majumderatax. Hosts vary from snails to mussels, but the mussels are mainly in the Northern Hemisphere in the Unionidae. The mantle mites appear to

be derived from the AfroEurAsian group of gill mites; however, Ampullariatax breaks out as a separate clade representing a separate divergence among the mantle mites. Pentatax is also somewhat separate from the remaining mantle mites and may represent a separate divergence. Mantle mites represent several somewhat distinctive clades within the group—each appears to support a distinct subgenus.

7. The gill mites form rather tight clades within the above-mentioned groups. In the Southern group, the 2 subgenera clearly separate.

8. In the American group, Australatax definitely appears to be the most ancient subgroup. Unionicolella falls within the Unionicolides; thus Unionicolella should be placed in synonomy. Atacella and Berezatax group together, but they are clearly separate from one another and the other subgenera in the analysis. Polyatacides and Ferradasatax are not included in the analysis.

9. In the AfroEurAsian group, considerable molecular analysis has been done on 3 subgenera, Unionicola, Wolcottatax and Dimockatax. Thus, we have an idea as to the molecular difference between these subgenera (Wu et al. 2008 and Edwards et al. 2010). Prasadatax falls right in the middle of these 3 subgenera—we have previously suggested that Prasadatax may represent the ancestral group for these subgenera and these 4 subgenera form an evolutionary unit of sorts. Interestingly, Unionicola, Wolcottatax and Dimockatax are found in anodontine mussels in North America.

10. In the AfroEurAsian group, Fulleratax is grouped with the African subgenera (Mutelicola, Chambardicola and Coelaturicola), which as a group are separate from the mites mentioned in 9. Iridinicola appears to be the least derived of this group and is somewhat separate in the tree.

Figure 8.4 Bayesian tree based on 139 morphological characters for representative species from 30 subgenera of Unionicola mollusk-associated mites. Numbers above branches represent posterior probability values. Letters indicate notable clades: A=Australian gill mites; B=gill mites, excluding those from Australia and mantle mites; C=gill mites from North and South America; D=African and Laurasian gill mites along with the mantle mites; E=mantle mites; F=African and Laurasian gill mites. Abbreviations in parentheses: G=gill mites; M=mantle mites; FS=free-

swimming mites; AFR=Africa; AUSTR=Australia; EUR=Europe; NA=North America; SA=South America. (figure from Edwards and Vidrine 2013)

Many of the subgenera are sorted based upon the female genital field structures. Van Reed prepared Plate 1 in order to show the variety of female genital fields in the genus. Female genital field morphology accounted for the first 20 character states considered in the list (see below). Additional plates (2-5) were prepared to show other major character states:

Plate 2: dorsal plates of varied species

Plate 3: pedipalps of varied species

Plate 4: first walking legs of varied species

Plate 5: modified walking legs of varied species.

Plate 1: female genital fields by category.

Plate 2: dorsal plates of varied species.

Plate 3: pedipalps of varied species.

Plate 4: first walking legs of varied species.

Plate 5: modified walking legs of varied species.

Appendix 6 (from Edwards and Vidrine 2013)

METHODS USED TO CONSTRUCT PHYLOGENETIC TREES AMONG

1) UNIONICOLA MITES AND 2) UNIONICOLA MOLLUSK MITES

Ingroup and Outgroup Taxa

Fifty-three of the 57 currently recognized Unionicola subgenera were used as terminal taxa to construct a morphological phylogeny for the genus, and were, in most cases, represented by their type species (Figures 8.2 and 8.3). Morphological data for mites from 30 subgenera were used to estimate the phylogeny among Unionicola mollusk mites (Figures 8.4 and 8.5). Seven of these subgenera are monotypic, thus a single species served as terminal taxa for these subgenera. Eight of the subgenera are relatively speciose, containing five or more (10.4+2.9SE, range=5-29) described species. For these subgenera, we used a minimum of four species for phylogenetic inference. The remaining 16 subgenera are represented by 2 to 4 species. In most instances, data from all species assigned to these subgenera were included in the analysis. Three subgenera of Unionicola (Downesatax. Ferradasatax and Polyatacides) were not included in this study because many of the character states of mites from these taxa were difficult to characterize. The water mite genus Neumania (Unionicolidae: Pionatacinae) was assigned as the outgroup taxon and was used to root the analysis. The subfamily Pionatacinae represents the sister taxon of the Unionicolinae. Mites of the genus Neumania are morphologically similar to the least-derived subgenus (e.g., Hexatax) of Unionicola (Vidrine 1996b) and a morphologically-based tree generated by Proctor and Wilkinson (2001) indicates that the genus Neumania is the sister taxon to mites of the genus Unionicola.

Character choice and coding

The character matrix used to construct the Unionicola tree (N=61 species from 53 subgenera) was comprised of 158 characters (156 morphological characters and 2 life history characters), whereas the mollusk-mite tree (N=90 species from 30 subgenera) was estimated based on 139 characters (137 morphological characters and 2 life history characters). These character state matrices are not provided here, but have been made available online:

1) Unionicola mite matrix: http://www.evansville.edu/majors/biology/downloads/Unionicola_character_matrix.pdf

2) Unionicola mollusk mite matrix: http://www.evansville.edu/majors/biology/downloads/Mollusk_character_Matrix.pdf .

Some of the characters that were used to reconstruct evolutionary relationships among Unionicola mites were based on those used either by Edwards and Vidrine (2006) or Wu et al., (2009) to address evolutionary relationships among mussel mites of North America and China, respectively. Additional characters, along with their states, were generated by reviewing the taxonomic literature for Unionicola, especially that which provided diagnoses of the genus and its subgenera, including Cook (1974), Vidrine (1980a, 1986e, 1996c and in this work). The characters (see the list provided below) used for phylogenetic reconstruction of Unionicola mites were based on features of female genital fields, male genital fields, pedipalps, pedipalp clawlets, venters with coxal plates, walking legs, tarsal claws, dorsums with plates, sexual dimorphisms of legs, body size, host associations and oviposition sites. Character states for Neumania, the outgroup taxon, were primarily obtained from descriptions by Cook (1974) and from specimens of Neumania sp. in Vidrine’s collection. We used two methods for coding character differences in our morphological and life history data, following the recommendations of Strong and Lipscomb (1999): 1) binary coding with inapplicable scenarios treated as “?”, and 2) ordered multistate character coding where absences were treated as a separate character. The sequence of states in binary code or transformation series were left unordered because it was, in many cases, difficult to determine a nested (ordered) set of synapomorphies based on outgroup comparison.

List of Characters and Character States

Asterisks (*) indicate those characters not used in reconstructing the mollusk mite trees (Figures 8.4 and 8.5).

Female genital fields

1. Female genital field with sclerotized acetabular plates containing acetabula: 0=yes; 1=no.

2. Number of pairs of acetabular plates: 0=absent; 1=2 pairs; 2=1 pair; 3=1 pair with sutures suggesting the presence of 2 pairs.

3. Acetabular plates appressed medially into a concise field: 0=no; 1=yes; 2=no, but plates appear to be derived (from ancestors with medially appressed plates).

4. Sclerotization of acetabular plates: 0=obviously sclerotized; 1=weakly sclerotized (well sclerotized only along the medial margins of the plates); 2=not sclerotized.

5. Genital field with obvious medial setae: 0=yes; 1=no.

6. Genital field with medial flaps: 0=no; 1=yes.

7. Number of pairs of acetabula on genital plates: 0=3-7; 1=8-12; 2=13-20; 3=>20.

8. Arrangement of acetabula on genital plates: 0=spread out evenly across the genital plates; 1=in clusters, except along the medial flaps; 2=in a line along the lateral margin of the genital plates.

9. Acetabula all the same size: 0=yes; 1=no.

10. Medial flaps in genital field: 0=absent; 1=present but inconspicuous; 2=present and conspicuous.

11. Location of medial flaps in genital field: 0=positioned centrally; 1=positioned anteriorly; 2=positioned posteriorly; ?=inapplicable, medial flaps absent.

12. Medial flaps modified into a spine-like, chitinized projection: 0=no; 1=yes, for those with 2 acetabular plates; 2=yes, on anterior acetabular plates only for those with 4 plates; ?=inapplicable, medial flaps absent.

13. Type of setae on the medial flaps: 0=no spinous setae; 1=at least one pair of spinous setae; ?=inapplicable, medial flaps absent.

14. Characteristics of medial setae on flaps on acetabular plates: 0=only hairlike setae found on acetabular flaps; 1=only spinous setae found on

acetabular flaps; 2=a mixture of hair-like and spinous setae found on acetabular flaps; ?=inapplicable, medial flaps absent.

15. Arrangement of seta in the anterior acetabular plate: 0=arranged in a row of 4 setae on the medial edge of flaps; 1=not arranged in a row of 4 setae on the medial edge of flaps; ?=inapplicable, no anterior plates present.

16. Medial flaps on acetabular plates: 0=small medial flaps with setae on 4 plates (as in Hexatax); 1=long medial flaps with setae on 4 plates (as in Hyricola); 2=flaps not as described above; ?= inapplicable, no flaps present.

17. Types of medial setae on acetabular plates (or on anterior acetabular plates if 4 plates are present): 0=hairlike; 1=thickened; 2=spinous; 3=mix of various setae, ?=inapplicable, medial setae absent.

18. Female genital field wider than it is long: 0=no; 1=yes but <3 times as wide as long; 2=yes, >3 times as wide as long).

19. Acetabula confined to acetabular plates: 0=no; 1=yes.

20. Female genital field with 4 near equal plates: 0=no; 1=yes, each with <7 pairs of acetabula; 2=yes, each with 7 or more pairs of acetabula.

21. Posterior glandularia (one on either side of genital field) on obvious cuplike structures: 0=yes; 1=reduced to very small to inconspicuous but still present; 2=absent.

Male genital fields

22. Male genital field with sclerotized acetabular plate(s) containing acetabula: 0=yes; 1=no.*

23. Sclerotization of acetabular plates: 0=obviously sclerotized; 1=weakly sclerotized (well sclerotized only along at least one of the margins of the plates); 2=not sclerotized.

24. Genital field with obvious medial setae: 0=yes; 1=no.

25. Number of pairs of acetabula: 0=3-7; 1=8-12; 2=13-20; 3=>20.

26. Arrangement of acetabula: 0=spread out evenly across the genital field; 1=in clusters; 2=in a line along the lateral margin of the genital field.

27. Acetabula all the same size: 0=yes; 1=no.

28. Location of the gonopore in the genital field: 0=central; 1=anterior; 2=posterior; 3=on the dorsum.

29. Male genital field wider than it is long: 0=no; 1=yes, <3 times as wide as long; 2=yes >3 times as wide as long).

30. Acetabula confined to acetabular plates: 0=no; 1=yes.

31. Male genital field extending onto the dorsum: 0=no; 1=yes.

32. Male genital field with 1 obvious seta or spine/side/plate: 0=no; 1=yes, in center of genital field; 2=yes, displaced posteriorly.

33. Male genital field with 2 obvious setae or spines/side/plate: 0=no; 1=yes, in center of genital field; 2=yes, located anteriorly; 3=yes, located posteriorly.

34. Number of setae on male genital field: 0=># (for numerous); 1=<# (for few (not numerous) and inconspicuous).

35. Arrangement of acetabula such that at least one acetabulum is out of linear arrangement if acetabula are in a line: 0—no, 1—yes, ?=inapplicable (acetabular arrangement not in a row or scattered).

36. Arrangement of acetabula: 0=not in a line along the margin of the plates, but spread out in the field; 1=in a line.

37. Male genital field with obvious sutures on plates on either side: 0=no; 1=yes.

Pedipalps and clawlets and chaetotaxy

38. Female pedipalps: 0=greater than half the length of the idiosoma;1=greater than one-quarter the length of the idiosoma; 2=less than one-quarter the length of the idiosoma.

39. Female palpal tarsus with ventral swelling containing setae: 0=no; 1=yes.

40. Female pedipalps swollen at the base (femur segment noticeably wider than other segments): 0=no; 1=yes.

41. Pedipalps sexually dimorphic: 0=no; 1=yes.

42. Shape of pedipalps: 0=subcylindrical; 1=dorsoventrally flattened.

43. Female longest palpal tarsal clawlet: 0=shorter than length of tarsus; 1=longer than length of tarsus.

44. Female palp tarsus longer than tibia: 0=no; 1=yes.

45. Female palpal tarsus greater than half the length of tibia: 0=yes; 1=no.

46. Female pedipalp tarsus tapered distally (toward a point) with clawlets not prominent: 0=yes; 1= no.

47. Female pedipalp clawlets (2 most obvious) near equal in size: 0=yes; 1=no; 2=only one clawlet present.

48. Female number of obvious tarsal clawlets: 0=3-4; 1=2; 2=1; 3=reduced to chitinous pustules.

49. Female palpal tibia with large, dorsal extension: 0=no; 1=yes.*

50. Female palpal tibia with a ventral protrusion containing setae: 0=yes; 1=no.

51. Female palpal tarsus fist-shaped (e.g., thicker distally than proximally): 0=no; 1=yes.*

52. Female at least one tarsal clawlet >2X length of tarsus: 0=no; 1=yes.

53. Chelicera fused medially: 0=yes; 1=no.*

54. Male pedipalp with large clawlike (sicklelike) clawlet much longer than tarsus: 0=no; 1=yes.*

Venter, coxal plate shapes and projections

55. Shapes of male and female coxal plates similar: 0=yes; 1=no.

56. Shape of posterior coxal group (coxal plates III and IV): 0=boxlike; 1=pointed posteriorly; 2=rounded posteriorly; 3=posterior border indistinct.

57. Female coxal plates fused into groups: 0=4 groups; 1=3 groups; 2=2 groups; 3=I group.

58. Male coxal plates fused into groups: 0=4 groups; 1=3 groups; 2=2 groups; 3=I group.

59. Female coxal plate anterior group (coxal plates I and II) with posterior projection: 0=yes; 1=no.

60. Female coxal plate posterior group (coxal plates III and IV) with posterior projection: 0=yes; 1=no.

61. Posterior projection of coxal plate anterior group (coxal plates I and II): 0= extending beyond the coxal plate III and IV suture; 1= extending to the suture between coxal plate III and IV; 2=extending to coxal plate III; ?=inapplicable, posterior projection absent.

62. Complete suture between coxal plates III and IV: 0=yes; 1= no.

63. Knob-like extensions on medial coxal plate I: 0=present; 1=absent.

64. Coxal plates groups with tooth-like lateral projections: 0=yes; 1=no.

65. Female medial edges of coxal plates III and/or IV apparent and well-sclerotized and not fused to the other posterior coxal plate group: 0=no; 1=yes.

66. Coxal plate IV elongated (>2x length of coxal plate III): 0=no; 1=yes.

67. Lateral gap between coxal plates III and IV: 0=no; 1=yes. 68. Chitinous anterior extension on coxal plate III: 0=absent; 1=present.

Legs and tarsal claws and chaetotaxy

69. Male and female leg I similar: 0=yes; 1=no.

70. Female genu of leg I with hairlike setae only: 0=no; 1=yes.

71. Female tibia of leg I with hairlike setae only: 0=no; 1=yes.

72. Female tarsus of leg I straight (not curved, bent, concave or arcuate or swollen at the base): 0=yes; 1=no.

73. Female tibia of leg I straight (not curved, bent, concave or arcuate or swollen at the base): 0=yes; 1=no.

74. Female tibia of leg I longer than genu: 0=no; 1=yes.

75. Female tibia of leg I longer than tarsus: 0=no; 1=yes.

76. Female number of large setae on genu: 0=2pairs; 1=1 pair; 2=0; 3=>2 pairs.

77. Female number of large setae on tibia: 0=2pairs; 1=1 pair; 2=0; 3=>2 pairs.

78. Female leg I with long, large, moveable, blunt setae on raised cuplike structures: 0=no, setae pointed and not raised on cuplike structures; 1=inapplicable; 2=yes; 3=no, setae pointed and raised on cuplike structures, ?=inapplicable, no large, long setae on leg I.

79. Female leg I genu, tibia, and tarsus exclusively with hairlike setae: 0=yes; 1=no.

80. Female leg I with telofemur, genu, and tibia all nearly as wide as long: 0=no; 1=yes.

81. Distal, dorsal, spoonlike setae over tarsal claws: 0=absent; 1= present.

82. Female distal, ventral, blunt setae on tibia of leg I: 0=absent; 1=present, setae smooth; 2=present, setae ornate.

83. Leg I distinctly wider than other walking legs: 0=yes; 1=no.

84. Female leg I with blunt setae on genu and tibia only: 0=no; 1=yes; ?=not applicable

85. Female leg I with one or more rows of at least 4 straight hairlike setae: 0=no; 1=yes, one row; 2=yes, two or more rows.

86. Female leg I longer than length of idiosoma: 0=yes; 1=no.

87. Female leg IV more than 2X length of idiosoma: 0=yes; 1=no.

88. Female leg IV with arcuate tarsus: 0=no; 1=yes.

89. Female leg IV with 2-3 long setae at distal end of genus and tibia: 0=yes; 1=no; 2=23 setae present but obviously reduced in length.

90. Male and female leg III similar: 0=yes; 1=no.

91. Male and female leg IV similar: 0=yes; 1=no.

92. Leg IV of female with conspicuous setae: 0=yes; 1=no.

93. Legs III-IV of both sexes with at least one row of four straight, hairlike setae on the genu and tibia: 0=no; 1=yes.

94. Female leg IV with large, blunt, dorsal, distal spines on telofemur and genu. 0=no; 1=yes.

95. Female leg I with dense, hairlike setae on dorsal surface of genu and tibia: 0=no; 1=yes.

96. Female leg IV with dense, hairlike setae on dorsal surface of telofemur, genu, and tibia: 0=no; 1=yes.

Sexual dimorphism of legs

97. Sexual dimorphism of leg I: 0=leg I of males without 2 large setae on telofemur; 1=Leg I of males with 2 large setae on telofemur.

98. Sexual dimorphism of leg I: 0=leg I equally setose in males and females; 1=leg I noticeably less setose in males than in females.

99. Sexual dimorphism of leg IV: 0=leg IV of males without bundles of setae on genu and tibia; 1=Leg IV of males with bundles of setae on genu and tibia.

100. Sexual dimorphism of leg IV: 0=leg IV of males without bundles of setae on telofemur, genu, and tibia; 1=Leg IV of males with bundles of setae on telofemur, genu, and tibia.

101. Sexual dimorphism of leg IV: 0=leg IV of males without bundles of setae on telofemur and genu; 1=Leg IV of males with bundles of setae on telofemur and genu.

102. Sexual dimorphism of leg IV: 0=leg IV of males without a large, blunt distal dorsal seta on telofemur and genu; 1=Leg IV of males with a large, blunt distal dorsal seta on telofemur and genu.

103. Sexual dimorphism of leg IV: 0=leg IV of males without large curved spines at distal end of genu; 1=Leg IV of males with large curved spines at distal end of genu.

104. Sexual dimorphism of leg IV: 0=tibia and tarsus of leg IV of males not grossly modified and tarsal claw obviously visible; 1=tibia and tarsus of leg IV of males grossly modified and tarsal claw not obviously visible.*

105. Sexual dimorphism of leg IV: 0=genu and tibia of leg IV of males without with large blunt spines; 1=genu and tibia of leg IV of males with large blunt spines, tibia noticeably short; 2=genu and tibia of leg IV of males with large blunt spines, tibia elongate.

106. Sexual dimorphism of leg IV: 0=leg IV of males without large blunt distal, dorsal, straight spine on genu; 1=leg IV of males with large blunt distal, dorsal, straight spine on genu.

107. Sexual dimorphism of leg IV: 0=leg IV of males without a series of short blunt serrated setae on ventral side of genu and tibia; 1=leg IV of males with a series of short blunt serrated setae on ventral side of genu and tibia.

108. Sexual dimorphism of leg IV: 0=claws of leg IV of males similar to females or bifid or as on all other legs; 1=claws of leg IV of males simple, whereas trifid on all other legs.*

109. Sexual dimorphism of leg IV: 0=leg IV of males without ventral indentation and stout setae on genu, tibia, and tarsus; 1=leg IV of males with ventral indentation and stout setae on genu, tibia, and tarsus.

110. Sexual dimorphism of leg IV: 0=leg IV of males without stout, curved setae at distal ends of telofemur, genu, and tibia; 1=leg IV of males with stout, curved setae at distal ends of telofemur, genu, and tibia.

111. Sexual dimorphism of leg IV: 0=leg IV of males without stout, ventral setae on telofemur, genu, and tibia; 1=leg IV of males with stout, ventral setae on telofemur, genu, and tibia.

112. Sexual dimorphism of leg IV: 0=leg IV of females without one or more rows of four straight setae on the genu and tibia; 1=leg IV of females with one or more rows of four straight setae on the genu and tibia.

Tarsal claws of walking legs

113. Tarsal claws of males and females similar: 0=yes; 1=no.*

114. All tarsal claws of walking legs: 0=finely bifid at tip; 1=simple; 2=deeply bifid; 3=other than above.

115. All tarsal claws similar: 0=yes; 1=no.

116. Female Tarsal claw of leg I different from those of other walking legs: 0=no; 1=yes, different from 2 other legs; 2=yes, different from one other leg.

117. Dorsal teeth on the tarsal claws pectinate: 0=no; 1=yes.

118. Ventral teeth on the tarsal claws pectinate: 0=no; 1=yes.*

119. Lateral teeth on the tarsal claws pectinate: 0=no; 1=yes.*

120. Tarsal claws noticeably large (at least as long as tarsus is wide at its midpoint): 0=no; 1=yes.

121. Tarsal claw of leg I of females bifid: 0=yes; 1=no.

122. Bifid tarsal claw of Leg I of females with equal prongs: 0=yes; 1=no, dorsal prong as long or longer than ventral prong; 2=no, ventral prong longer than dorsal prong; ?=inapplicable, tarsal claws of leg I not bifid.

123. Tarsal claws of Leg II of females bifid: 0=yes; 1=no.

124. Bifid tarsal claw of Leg II of females with equal prongs: 0=yes; 1=no, dorsal prong as long or longer than ventral prong; 2=no, ventral prong longer than dorsal prong; ?=inapplicable, tarsal claws of leg II not bifid.

125. Tarsal claws of Leg III of females bifid: 0=yes; 1=no.

126. Bifid tarsal claw of Leg III of females with equal prongs: 0=yes; 1=no, dorsal prong as long or longer than ventral prong; 2=no, ventral prong longer than dorsal prong; ?=inapplicable, tarsal claws of leg III not bifid.

127. Tarsal claw of leg IV of females bifid: 0=yes; 1=no.

128. Bifid tarsal claw of Leg IV of females with equal prongs: 0=yes; 1=no, dorsal prong as long or longer than ventral prong; 2=no, ventral prong longer than dorsal prong; ?=inapplicable, tarsal claws of leg IV not bifid.

129. Tarsal claw of leg I of females trifid: 0=no; 1=yes.*

130. Tarsal claw of leg IV of females trifid: 0=no; 1=yes.

131. Tarsal claws on at least one leg appearing as an inverted spoon with serrated edge: 0=no; 1=yes.*

132. Tarsal claws of leg I retractable: 0=no; 1=yes.

133. Tarsal claws of first 3 pairs of legs of males obviously bifid, last leg simple: 0=no; 1=yes.*

134. Female tarsal claws of leg I bifid and ‘cartoonish’: 0=no; 1=yes.

Dorsum plates and chaetotaxy

135. Male and female dorsal surfaces/plates similar: 0=yes; 1=no.

136. Male dorsal apodemes apparent: 0=no; 1=yes.

137. Female dorsal apodemes apparent: 0=no; 1=yes.

138. Male dorsal plates obvious: 0=no; 1=yes.

139. Female dorsal plates obvious: 0=no; 1=yes.

140. Male dorsum with 4 small plates in the anterior portion: 0=no; 1=yes.

141. Female dorsum with 4 small plates in the anterior portion: 0=no; 1=yes.

142. Male dorsal plates cover more than half the dorsum or nearly so: 0=no; 1=yes.

143. Female dorsal plates cover more than half the dorsum or nearly so: 0=no; 1=yes.

144. Number of dorsal plates on males: 0=none; 1=1 plate; 2=divided into 2 or more plates.

145. Number of dorsal plates on females: 0=none; 1=1 plate; 2=divided into 2 or more plates.

146. Dorsal plates of males with associated apodemes: 0=no; 1=yes.

147. Dorsal plates of females with associated apodemes: 0=no; 1=yes.

148. Dorsal plates of males with obvious spinous setae: 0=no; 1=yes.

149. Dorsal plates of females with obvious spinous setae: 0=no; 1=yes.

Body size

150. Female Body size: 0=<400µm; 1=400-999µm; 2=1mm-2mm; 3=>2mm.

151. Body white in color: 0=no; 1=yes.

152. Body elongate with a projecting posterior (obvious cauda in males): 0=no; 1=yes.

Larval characters

153. Shape of anal plate of larvae: 0=circular or oval; 1=triangular (wider than long).*

154. Number and location of setae on anal plate of larvae: 0=2 setae arising anterior and two posterior; 1=4 setae arising along posterior edge.*

155. Larvae brown in color or transparent: 0=yes; 1=no.*

156. Larvae with posterior pair of setae arising wide apart (distance equal to the length of a seta): 0=no; 1=yes.*

Host-associations

157. Lifestyle: 0=free-swimming mite; 1=sponge-associated mite; 2=snail-associated mite; 3=mussel-associated mite.

Egg deposition

158. Oviposition: 0=in sponges; 1=in molluscan mantle tissue; 2=in molluscan gill tissue; 3=unknown; ?=inapplicable, free-swimming mite.

Phylogenetic analyses

Characters were analyzed using Bayesian optimality criteria. All analyses treated binary and multistate characters as unweighted and unordered. Bayesian analysis was conducted with default priors and the Markov k model with a gamma (Mk+G) distribution (Lewis 2001) using MrBayes 3.1 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). Two analyses ran in parallel each with four chains of five million generations and the posterior distribution was sampled every 5000 generations. We concluded that the analysis had reached stationarity when the average standard deviation of split frequencies was less than 0.01, evidence of chain swapping was sufficient, and the potential scale reduction factors (PSRF) were near one. An appropriate burnin was assessed by a plot of log likelihoods viewed in the program Tracer v1.4 (Rambaut and Drummond 2007) and was discarded before summarizing model parameters and tree statistics using the sump (burnin=500) and sumt (burnin=500) commands in MrBayes.